Molecular and functional characterization of olfaction genes in tsetse flies

描述（由申请人提供）： 这项研究的总体目标是提供采采蝇媒介中介导其嗅觉过程的分子过程的信息，使它们能够找到宿主和其他生存和繁殖的关键资源。这些目标将通过在肯尼亚具有医学和兽医重要性的选定采采蝇物种（Glossina pallidipes、Glossina austeni 和 Glossina fuscipes fuscipes）中探索和验证嗅觉响应基因来实现。我们提出了一种综合和跨学科的方法：1）确定辛巴山的同域 Glossina pallidipes 和 Glossina austeni 以及肯尼亚姆比塔的 Glossina fuscipes fuscipes 嗅觉响应基因的差异；2）确定辛巴山的同域 G. pallidipes 和 G. austeni 以及 G. f. 的嗅觉响应基因的功能作用。肯尼亚姆比塔地区的fuscipes。我们假设这些果蝇中假定的嗅觉基因是直系同源的、差异表达的并且显着影响果蝇对不同气味的反应。所选采采蝇的成虫将从各自的地点取样，并从头部组织中提取 RNA。我们将开展一个发现项目，利用下一代测序技术来确定不同物种之间表现出的全局转录组水平的异质性。从果蝇肠道内容物中获得的 DNA 将使用 COI 标记进行 PCR 扩增，以确定生命条形码数据系统 (BOLD) 数据库中的假定宿主物种。在对不同物种的转录组进行比较分析后，我们将通过 qPCR 在体外验证选定的一组推定嗅觉相关基因的存在和表达谱。将通过 RNAi 基因沉默方法结合电触角图和风洞技术进一步研究选定的假定嗅觉基因的功能作用，以确定这些基因在调节果蝇生理和行为中的作用。嗅觉相关基因的综合信息及其在调节采采蝇嗅觉行为中各自的功能作用的可用性将有助于进一步的研发，以利用这些基因来控制采采蝇媒介。这项研究将主要在肯尼亚国际昆虫中心进行 生理生态学和肯尼亚农业研究所 - 锥虫病研究中心与 Paul Mireji 合作，配套资助为全球传染病 (GID) D43 TW007391 (08/01/2008-03/31/2013)。

DESCRIPTION (provided by applicant): The overall goal of this study is to provide information on molecular processes in tsetse vectors that mediate their olfactory processes, enabling them to locate their hosts and other critical resources for their survival and reproduction. The goals will be achieved by prospecting for and validating olfaction responsive genes in selected tsetse species (Glossina pallidipes, Glossina austeni and Glossina fuscipes fuscipes) of medical and veterinary importance in Kenya. We propose an integrated and interdisciplinary approach to: 1) Determine differences in olfactory responsive genes in sympatric Glossina pallidipes and Glossina austeni in Shimba hills and Glossina fuscipes fuscipes in Mbita in Kenya and 2) Determine functional roles of olfactory responsive genes in sympatric G. pallidipes and G. austeni in Shimba hills and G. f. fuscipes in Mbita districts in Kenya. We hypothesize that putative olfactory genes among these flies are orthologuous, differentially expressed and significantly influence responses of the flies to different odors. Adults of the selected tsetse species will be sampled from respective sites and RNA will be extracted from head tissues. We will undertake a discovery project by using next generation sequencing technologies to determine global transcriptome-level heterogeneities exhibited between the different species. The DNA obtained from the gut contents of flies will be subjected to PCR amplification with COI marker to determine the putative host species from Barcode of Life Data systems (BOLD) database. Following the comparative transcriptome analysis from the different species we will in vitro validate the presence and expression profiles of select set of putative olfaction related genes through qPCR. The functional role(s) of selected putative olfaction genes will be further investigated through RNAi gene silencing methodologies in conjunction with electro-antennogram and Wind-Tunnel techniques to establish the roles of these genes in modulating the physiology and behavior of the flies. Availability of comprehensive information on olfaction related genes, and their respective functional roles in modulating tsetse olfactory behavior will facilitate further R&D to exploit these genes to control tsetse fly vectors. This research will be done primarily in Kenya at International Centre of Insect Physiology Ecology and Kenya Agricultural Research Institute - Trypanosomiasis Research Center in collaboration with Paul Mireji with companion grant being Global Infectious Diseases (GID) D43 TW007391 (08/01/2008- 03/31/2013).